Project Summary/Abstract?Applied Research Component The goal of the Applied Research Component of the Caenorhabditis Genetics Center (CGC) is to enhance the CGC strain collection. The CGC aims to curate strains of importance to the biomedical research field, including null mutations in all protein coding genes as well as ncRNAs, certain transgenic strains, and genetic tools. The C. elegans molecular genetic tool-kit has a deep foundation built upon identified mutations in many thousands of genes, genome-wide RNAi libraries, easy and efficient methods of transgenesis, well developed and rapidly evolving methods for genome modification using Crispr-Cas9 techniques, mechanisms for tissue-specific gene activation and protein destruction, and others. The applied research component will use Crispr-Cas9 genome modification methods to expand the strain collection. In Aim 1, we propose to improve the C. elegans genetic balancer collection. Genetic balancers are chromosomal rearrangements that act as crossover suppressors. They are essential useful tools for working efficiently with lethal or sterile mutations, allowing them to be maintained easily as heterozygotes. They also facilitate genetic crosses of mutants with subtle phenotypes. A particularly useful class of crossover suppressors encompasses intrachromosomal inversions that are also marked so as to allow different progeny classes to be readily identified. Recently developed Crispr-Cas9 strategies allow creation of targeted chromosomal rearrangements allowing us to make designer inversions. We will build a set of inversion-based crossover suppressors that fully covers the C. elegans genome, marked with red or green-fluorescent reporters. In Aim 2, to support the goal of curating a null mutation in every gene, the CGC will make a collection of mutations in miRNA genes. microRNAs (miRNAs) are small regulatory RNAs, first discovered through basic research in C. elegans, that have profoundly changed our understanding of eukaryotic gene regulation in processes including development, metabolism, stem cell maintenance, and cancer; with demonstrated roles in human development and disease, they are being developed as diagnostic as well as therapeutic tools. To facilitate community research on the functions of this important gene class, we will make deletion alleles of this remaining set of miRNAs, again using established Crispr-Cas9 methods. All strains generated in this work will be curated by the CGC and immediately available through the CGC website.